Sim2 polypeptides and polynucleotides and uses of each in diagnosis and treatment of ovarian, breast and lung cancers

ABSTRACT

A method of diagnosing predisposition to, or presence of ovarian cancer, breast cancer and/or lung cancer in a subject is provided. The method comprises determining a level of SIM2 in a lung tissue, breast tissue and/or ovarian tissue of the subject, the level being correlatable with predisposition to, or presence or absence of the ovarian cancer, breast cancer and/or lung cancer, thereby diagnosing predisposition to, or presence of ovarian cancer, breast cancer and/or lung cancer in the subject.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to SIM2 polypeptides and polynucleotidesand to methods of diagnosing and treating ovarian, breast and lungcancers.

Lung cancer is the primary cause of cancer death among both men andwomen in the United States. The five-year survival rate among all lungcancer subjects, regardless of the stage of disease at diagnosis, isonly 13%. This contrasts with a five-year survival rate of 46% amongcases in which the disease is still localized. However, only 16% of lungcancers are diagnosed at a stage prior to spread of the disease.

Early detection is difficult since clinical symptoms are often notobserved until the disease has reached an advanced stage. Currently,diagnosis is aided by the use of chest x-rays, analysis of the type ofcells contained in sputum and fiberoptic examination of the bronchialpassages. Treatment regimens are determined by the type and stage of thecancer, and include surgery, radiation therapy and/or chemotherapy. Inspite of considerable research into therapies for the disease, lungcancer remains difficult to treat.

Breast cancer is the most common form of cancer in women, and can alsobe diagnosed in men. Over 200,000 new breast cancer cases are diagnosedeach year in the United States. In the U.S. today, there are more thantwo million breast cancer survivors, and every woman is at risk.

Molecular biomarkers for breast cancer are of several types. Riskbiomarkers are those associated with increased cancer risk and includemammographic abnormalities, proliferative breast disease with or withoutatypia, family clustering and inherited germ-line abnormalities.Surrogate endpoint biomarkers are tissue, cellular or molecularalterations that occur between cancer initiation and progression. Thesebiomarkers are utilized as endpoints in short-term chemopreventiontrials. Prognostic biomarkers provide information regarding outcomeirrespective of therapy, while predictive biomarkers provide informationregarding response to therapy. Candidate prognostic biomarkers forbreast cancer include elevated proliferation indices such as Ki-67 andproliferating cell nuclear antigen (PCNA); estrogen receptor (ER) andprogesterone receptor (PR) overexpression; markers of oncogeneoverexpression such as c-erbB-2, TGF-a and EGFR; indicators of apoptoticimbalance including overexpression of bcl-2 and an increased bax/bcl-2ratio; markers of disordered cell signaling such as p53 nuclear proteinaccumulation; alteration of differentiation signals such asoverexpression of c-myc and related proteins; loss of differentiationmarkers such as TGF-b II receptor and retinoic acid receptor; andalteration of angiogenesis proteins such as VEGF overexpression [Beenken(2002) Minerva Chir. 57:437-48].

Availability of molecular biomarkers for breast cancer willsubstantially increase diagnostic capabilities and enable thedevelopment of prognostic indices, which combine the predictive power ofindividual molecular biomarkers with specific clinical and pathologicfactors.

Ovarian cancer, is a significant health problem for women in the UnitedStates and world wide. Although advances have been made in detection andtherapy of this cancer, no vaccine or other universally successfulmethod for prevention or treatment is currently available. Management ofthe disease currently relies on a combination of early diagnosis andaggressive treatment, which may include one or more of a variety oftreatments such as surgery, radiotherapy, chemotherapy and hormonetherapy. The course of treatment for a particular cancer is oftenselected based on a variety of prognostic parameters, including ananalysis of specific tumor markers. However, the use of establishedmarkers often leads to a result that is difficult to interpret, and highmortality continues to be observed in many cancer subjects.

Thus, there remains a need for a practical method of diagnosing lungcancer, breast cancer and ovarian cancer as close to inception aspossible. In order for early detection to be feasible, it is importantthat specific markers be found and their sequences elucidated.

The Drosophila single minded (sim) gene is the master regulator of fruitfly meurogenesis [Thomas (1988); Mambu (1991)]. SIM protein is atranscription factor containing a basic helix-loop-helix (bHLH) motif,two PAS (PER/ARNT/SIM) domains, and an HST (HIF-α/SIM/TRH) domain [Mambu(1991); Isaac and Andrew (1996)].

Two mouse homologs of the sim gene (i.e., Sim1 and Sim2) were cloned.Sim1 maps on mouse chromosome 10 and Sim2 on mouse chromosome 16 in aregion of synteny with HC21 [Fan et al. (1996) Mol. Cell. Neurosci.7:1-16]. Both mouse Sim1 and Sim2 genes are expressed early [Sim2 fromembryonic day 8.0 (E8.0) and Sim1 from day 9.0 (E9.0)] in developingforebrain [Ema et al. (1996) Mol. Cell. Biol. 16:5865-5875; Fan et al.(1996) supra; Moffett et al. (1996) Genomics 35:144-155; Yamaki et al.(1996) Genomics 35:136-143] and outside the central nervous system(CNS), in somites, mesonephric duct, and foregut (SIM1), in facial andtrunk cartilage, trunk muscles (Sim2), and in the developing kidney(SIM1 and SIM2) [Dahmane et al. (1995) Proc. Natl. Acad. Sci.92:9191-9195; Ema et al. (1996) supra; Fan et al. (1996) supra; Moffettet al. (1996) supra].

In adult mouse, both SIM1 and SIM2 are expressed in kidney and skeletalmuscles, whereas Sim2 is also expressed in the lung (Ema et al. (1996)Biochem. Biophys. Res. Commun. 218:588-594; Moffett et al. (1996)supra].

Recently, the cloning of the cDNAs for two human homologs (SIM1 andSIM2) of the Drosophila sim gene, and mapping of SIM1 to chromosome6q16.3-q21 have been reported [Chrast (1997) Genome Research 7:615-624and Chen et al. (1995)]. Northern blot analyses indicated thetranscription of several mRNA transcripts from the SIM2 gene, includingthose of 2.7, 3, 4.4 and 6 kb. The multiple mRNAs may be products ofalternative splicing, overlapping transcription, or differentutilization of 5′ or 3′ untranslated sequences. At least two differentforms of the human SIM2 gene have been characterized. The long form(GenBank Accession No. U80456; SEQ ID NO: 7 is 3921 bp and codes for aprotein of 667 amino acid with an apparent molecular weight of 74 kD.The short-form (GenBank Accession No. U80457; SEQ ID NO: 8) is 2859 bpand codes for a protein of 570 amino acid with an apparent molecularweight of 64 kD.

Human SIM-1 and SIM-2, in combination with ARNT, attenuate transcriptionfrom the hypoxia-inducible erythropoietin (EPO) enhancer during hypoxia.SIM protein levels decrease with hypoxia treatment, suggesting anegative feedback mechanism. Upregulation and activation of HIF-1α isconcomitant with attenuation of SIM activities [Woods S L., Whitelaw ML., J. Biol. Chem 2002; 277:10236-43].

PCT Application No. WO 02/12565 discloses the use of SIM2 as a markerand possible therapeutic target for specific types of cancer. Increasedexpression of SIM2 in specific cancers including colon, prostate andpancreas tumors as compared to normal tissues was observed. Inaccordance, a similar pattern of expression was found by DeYoung andco-worlers [Proc. Natl. Acad. Sci. (2003) 100:4760-5 and Anticancer Res.(2002) 22(6A):3149-57]. Interestingly, neither of the above-publicationsshowed an elevated expression of SIM2 in ovarian, lung and breasttumors.

While reducing the present invention to practice the present inventorsuncovered that elevated levels of SIM2 are associated with ovarian,breast and lung tumors, thus showing for the first time that SIM2 canalso be used as a marker and possible therapeutic target for ovarian,breast and lung tumors.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of diagnosing predisposition to, or presence of ovarian cancer,breast cancer and/or lung cancer in a subject, the method comprisingdetermining a level of SIM2 in a biological sample obtained from thesubject, the level being correlatable with predisposition to, orpresence or absence of the ovarian cancer, breast cancer and/or lungcancer, thereby diagnosing predisposition to, or presence of ovariancancer, breast cancer and/or lung cancer in the subject.

According to further features in preferred embodiments of the inventiondescribed below, the biological sample is a tissue sample and/or a bodyfluid sample.

According to still further features in the described preferredembodiments the tissue sample is selected from the group consisting ofan ovarian tissue, a lung tissue and a breast tissue.

According to another aspect of the present invention there is provided amethod of treating ovarian cancer, breast cancer and/or lung cancer in asubject, the method comprising downregulating expression or activity ofSIM2 in a lung tissue, breast tissue and/or an ovarian tissue, therebytreating the ovarian cancer, breast cancer and/or lung cancer in thesubject.

According to still further features in the described preferredembodiments the SIM2 is selected from the group consisting of SEQ IDNOs: 1, 2, 3, 7, 8 and 9.

According to still further features in the described preferredembodiments downregulating expression or activity of the SIM2 iseffected by administering to the subject:

-   -   (a) a molecule which binds SIM2;    -   (b) an enzyme which cleaves SIM2;    -   (c) an antisense polynucleotide capable of specifically        hybridizing with an mRNA transcript encoding SIM2;    -   (d) a ribozyme which specifically cleaves SIM2 transcripts;    -   (e) a non-functional analogue of at least a catalytic or binding        portion of SIM2;    -   (f) a molecule which prevents SIM2 activation or substrate        binding;    -   (g) an siRNA molecule capable of inducing degradation of SIM2        transcripts;    -   (h) a DNAzyme which specifically cleaves SIM2 transcripts or        DNA; and    -   (i) a molecule which promotes a SIM2-specific immunogenic        response.

According to still further features in the described preferredembodiments the molecule which binds SIM2 is an antibody or antibodyfragment capable of specifically binding the SIM2.

According to yet another aspect of the present invention there isprovided use of an agent capable of downregulating SIM2 expression oractivity for the treatment of ovarian, breast and/or lung cancer.

According to still further features in the described preferredembodiments the agent capable of downregulating SIM2 activity is anantibody or antibody fragment.

According to still further features in the described preferredembodiments the agent capable of downregulating SIM2 expression oractivity is an oligonucleotide.

According to still further features in the described preferredembodiments the oligonucleotide is a single or double strandedpolynucleotide.

According to still further features in the described preferredembodiments the oligonucleotide is at least 17 bases long.

According to still further features in the described preferredembodiments the oligonucleotide is hybridizable in either sense orantisense orientation.

According to still another aspect of the present invention there isprovided use of a SIM2 detecting agent for detecting ovarian, breastand/or lung cancer.

According to still further features in the described preferredembodiments the agent for detecting ovarian, breast and/or lung canceris an oligonucleotide.

According to still further features in the described preferredembodiments the agent for detecting ovarian, breast and/or lung canceris an antibody or antibody fragment.

According to still further features in the described preferredembodiments the agent for detecting ovarian, breast and/or lung canceris coupled to a detectable moiety selected from the group consisting ofa chromogenic moiety, a fluorogenic moiety, a radioactive moiety and alight-emitting moiety.

According to an additional aspect of the present invention there isprovided an article-of-manufacture comprising a packaging material and acomposition identified for treating ovarian, breast and/or lung cancerbeing contained within the packaging material, the compositionincluding, as an active ingredient, an agent capable of downregulatingSIM2 expression or activity.

According to still an additional aspect of the present invention thereis provided an isolated polynucleotide comprising a nucleic acidsequence encoding a polypeptide being at least 80% homologous to SEQ IDNO: 39, 46 or 41 as determined using the BestFit software of theWisconsin sequence analysis package, utilizing the Smith and Watermanalgorithm, where the gap creation equals 8 and gap extension penaltyequals 2.

According to still further features in the described preferredembodiments the polypeptide is as set forth in SEQ ID NO: 39, 40 or 41.

According to a further aspect of the present invention there is providedan isolated polynucleotide comprising a nucleic acid sequence being 80%identical to SEQ ID NO: 39, 40 or 41, as determined using the BestFitsoftware of the Wisconsin sequence analysis package, utilizing the Smithand Waterman algorithm, where gap weight equals 50, length weight equals3, average match equals 10 and average mismatch equals −9.

According to still further features in the described preferredembodiments the nucleic acid sequence is as set forth in SEQ ID NO: 2 or3.

According to yet a further aspect of the present invention there isprovided an isolated polynucleotide as set forth in SEQ ID NO: 2 or 3.

According to still a further aspect of the present invention there isprovided a nucleic acid construct comprising the isolatedpolynucleotide.

According to still a further aspect of the present invention there isprovided an isolated polypeptide as set forth in SEQ ID NO: 39, 40 or41.

According to still a further aspect of the present invention there isprovided a method of diagnosing predisposition to, or presence of cancerin a subject, the method comprising determining a level of SEQ ID NO: 2and/or 3 in a biological sample obtained from the subject, wherein thebiological sample is suspected of being a cancerous tissue or associatedwith the cancerous tissue and whereas the level being correlatable withpredisposition to, or presence or absence of the cancer, therebydiagnosing predisposition to, or presence of cancer in the subject.

According to still further features in the described preferredembodiments the determining level of the SEQ ID NO: 2 and/or 3 iseffected at an mRNA level.

According to still further features in the described preferredembodiments the determining level of the SEQ ID NO: 2 and/or 3 iseffected at a protein level.

According to still further features in the described preferredembodiments the determining level of the SEQ ID NO: 2 and/or 3 iseffected at a gene amplification level.

According to still a further aspect of the present invention there isprovided a method of treating cancer in a subject, the method comprisingdown-regulating expression or activity of SEQ ID NO: 2 and/or 3 in acancerous tissue, thereby treating the cancer in the subject.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing SIM2 polypeptides andpolynucleotides encoding same which can be used to diagnose and treatovarian and lung cancers.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic illustration showing the genomic organization ofSIM2 expression products. Boxes designate exons and lines designateintrons. Arrow heads designate primers.

FIG. 2 is a histogram showing SIM2 expression in normal andtumor-derived lung samples as determined by real time PCR using SEQ IDNO: 20, normalized to the housekeeping gene RPS27A (SEQ ID NO: 23). Twoindependent experiments are shown.

FIG. 3 is a histogram showing SIM2 expression in normal and tumorderived colon samples as determined by real time PCR using a SIM2derived fragment (SEQ ID NO: 20).

FIG. 4 is a histogram showing SIM2 long and short transcript expressionin normal and tumor-derived lung samples as determined by real time PCRusing a SIM2 derived fragment (SEQ ID NO: 9) corresponding tocoordinates 232-404 of SEQ ID Nos 7 and 8. Expression of SIM-2 derivedfragment (SEQ ID NO: 9) was normalized to the expression of RPS27A (SEQID NO: 23) housekeeping gene. Two independent experiments are shown.

FIG. 5 is a histograms depicting SIM2 expression as in FIG. 4 on a 0-200scale.

FIG. 6 is a histogram showing SIM2 long and short transcript (SEQ IDNOs: 7 and 8) expression in normal and tumor derived lung samples asdetermined by real time PCR using a SIM2 derived fragment (SEQ ID NO:9). Expression of SEQ ID NO: 9 was normalized to the expression ofRPS27A (SEQ ID NO: 23) housekeeping gene.

FIG. 7 is a histogram depicting SIM2 expression as in FIG. 6 on a 0-200scale.

FIG. 8 is a histogram showing SIM2 expression in normal and tumorderived ovarian samples as determined by real time PCR using a SIM2derived fragments (SEQ ID NOs: 9 and 20). Expression was normalized tothe averaged expression of three housekeeping genes PBGD (SEQ ID NO:32), ATP-6-syn (SEQ ID NO: 26) and 18s ribosomal RNA (SEQ ID NO: 29).

FIG. 9 is a histogram showing SIM2 long variant expression in normal andtumor derived ovary samples as determined by real time PCR using a SIM2derived fragment (SEQ ID NO: 18). Expression was normalized to theaveraged expression of two housekeeping genes PBGD (SEQ ID NO: 32) andHPRT1 (SEQ ID NO: 35).

FIG. 10 is a histogram showing SIM2 long variant expression in normaland tumor derived lung samples as determined by real time PCR using aSIM2 derived fragment (SEQ ID NO: 18). Expression was normalized to theaveraged expression of three housekeeping genes SDHA (SEQ ID NO: 38),RPS27A (SEQ ID NO: 23) and PBGD (SEQ ID NO: 32).

FIG. 11 is a histogram showing SIM2 short variant expression in normaland tumor derived ovarian samples as determined by real time PCR using aSIM2 derived fragment (SEQ ID NO: 19). Expression was normalized to theaveraged expression of two housekeeping genes PBGD (SEQ ID NO: 32) andHPRT1 (SEQ ID NO: 35).

FIG. 12 is a photomicrograph showing the expression of SIM2 long variantin normal and tumor derived breast samples as determined by RT-PCR usinga SIM2 derived fragment (SEQ ID NO: 18).

FIG. 13 is a photomicrograph showing the expression of SIM2 shortvariant in normal and tumor derived breast samples as determined byRT-PCR using a SIM2 derived fragment (SEQ ID NO: 19).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of SIM2 polypeptides and polynucleotidesencoding same, which can be used to diagnose and treat ovarian and lungcancers.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Lung cancer and ovarian cancer are deadly diseases for which treatmentis currently limited. It is well established that early detection ofprimary, metastatic and recurrent diseases can significantly impact theprognosis of individuals suffering from lung cancer and ovarian cancer.

Current methods for early detection of ovarian cancer involvetransabdominal/transvaginal ultrsonography and a blood test for thetumor marker CA-125. While ultrasound screening is often misleadingsince most enlarged ovaries result from benign cysts, the use of thetumor marker CA-125 in diagnosing ovarian cancer is limited due to ahigh false-positive rate.

Early detection of lung cancer is currently effected using chest X-raysand sputum cytology. Since these methods do not have any effect onmortality, detection still may be too late to affect the natural causeof the disease.

The Drosophila single minded (SIM) gene is the master regulator of fruitfly meurogenesis [Thomas (1988); Mambu (1991)]. SIM protein is atranscription factor containing a basic helix-loop-helix (bHLH) motif,two PAS (PER/ARNT/SIM) domains, and an HST (HIF-α/SIM/TRH) domain [Mambu(1991); Isaac and Andrew (1996)]. The native human SIM2 gene has beencloned and multiple mRNA products of the gene have been found byNorthern blot analyses.

SIM2 has been previously associated with colon, pancreatic and prostatecancers but not with lung and ovarian cancers (PCT Application No.WO02/12565).

While reducing the present invention to practice the present inventorsuncovered, for the first time, that elevated levels of SIM2 are presentin ovarian, breast and lung tumors, thus providing evidence that thisgene can be utilized as a diagnostic marker for ovarian, breast and lungtumors, or can serve as a basis for a therapeutic agent for treatingsuch tumors.

Thus, according to one aspect of the present invention there is provideda method of diagnosing predisposition to, or presence of ovarian cancer,breast cancer and/or lung cancer in a subject.

As used herein the term “predisposition” refers to a latentsusceptibility to ovarian, breast and/or lung cancer, which may lead,under certain conditions, to the formation of ovarian, breast and/orlung cancer.

As used herein the phrase “ovarian cancer” refers to epithelial tumors(i.e., carcinomas) and non-epithelial tumors (e.g., stroma cell and germcell tumors of the ovary).

As used herein the phrase “breast cancer” refers to non-invasive andinvasive tumors of the breast including Lobular Carcinoma In Situ(LCIS), Ductal carcinoma, Ductal Carcinoma In Situ (DCIS) and CarcinomaIn Situ.

As used herein the phrase “lung cancer” refers to cancers of the lungincluding small cell lung cancer and non-small cell lung cancer.

The method, according to this aspect of the present invention iseffected by determining a level of SIM2 in a biological sample which isobtained from the subject thereby diagnosing predisposition to, orpresence of ovarian cancer, breast cancer and/or lung cancer in thesubject.

As used herein “a biological sample” “refers to a sample of tissue(e.g., breast, lung, ovary) or fluid isolated from a subject, includingbut not limited to, for example, plasma, serum, spinal fluid, lymphfluid, the external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, milk, blood cells, tumors, organs,and also samples of in vivo cell culture constituents.

As used herein, the term “level” refers to expression of SIM2 RNA and/orprotein or SIM2 DNA copy number.

As is further described hereinbelow and in the examples section whichfollows, the present inventors have shown that levels of SIM2 beyondthose found in normal tissue correlate with predisposition to, orpresence or absence of the ovarian cancer, breast cancer and/or lungcancer providing evidence that this gene can serve as a marker forbreast cancer, lung cancer and ovarian cancer.

As used herein SIM2 refers to a SIM2 gene as set forth in sequencecoordinates 34649835-34700062 of chromosome 21q22.13, expressionproducts of the SIM2 gene as well as, fragments and variants thereof.

As used herein the term “variants” refers to splice variants and allelicvariants of SIM2.

The phrase “splice variant” refers to alternative forms of RNAtranscribed from a SIM2 gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

The phrase “allelic variant” refers to two or more alternative forms ofa SIM2 gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequence. The term allelic variant is also used herein todenote a protein encoded by an allelic variant of a gene.

Examples of SIM2 expression products (i.e., splice variants) are setforth in SEQ ID NOs: 7 (GenBank Accession No. U80456) and 8 (GenBankAccession No. U80457), which correspond to the long form and short formof SIM2, respectively.

It will be appreciated that while reducing the present invention topractice additional SIM2 splice variant have been discovered by thepresent inventors. These are set forth in SEQ ID NOs: 2 and 3, furtherdescription of which is provided hereinbelow and in FIG. 1 and Table 1,below.

Numerous well known tissue or fluid collection (e.g., sputum collection)methods can be utilized to collect the biological sample from thesubject in order to determine the level of SIM2 DNA, RNA and/orpolypeptide of the subject.

Tissue biopsy can be utilized to collect a tissue sample from lungtissue, breast tissue and/or ovarian tissue. Methods of performing lung,breast and ovarian biopsies are well known in the art.

Typically, an ovarian biopsy is effected using fine-needle aspiration(FNA), which is an important diagnostic tool in gynecology. Its mainrole is in diagnosis of advanced and recurrent gynecologic malignancies.The technique uses a small-gauge needle to aspirate a lesion forcytologic analysis, sometimes with the aid of radiographic imaging.

A number of approaches for performing breast biopsies are known in theart.

A Stereotactic Needle Biopsy is a relatively novel approach, which isemployed when the physician cannot feel the lump that was found on amammogram. A Stereotactic Biopsy uses mammographic images and computertechnology and combines them to determine the exact location of anabnormality to obtain a sample of breast tissue. In this way,non-surgical techniques are used to determine whether an abnormality iscancerous. This procedure is equally accurate as surgical biopsy withoutthe scar and anesthetic risks of surgery. The patient lies facedown on aspecial table with an opening for the breast. Using special equipment,the breast is compressed similar to a mammogram but with lesscompression, and then x-rayed from several angles. The data is enteredinto a computer, and then a radiologist inserts a biopsy needle into thelump.

Fine Needle Aspiration involves inserting a very fine, hollow needleinto a cyst to remove some fluid or tissue.

Core Needle Biopsy utilizes a larger needle inserted into the breastmass to remove a tissue for examination.

Ductal Lavage is a relatively new, minimally invasive FDA approvedprocedure being used for women who are considered at high risk ofdeveloping breast cancer. The doctor inserts a catheter into a milk ductand withdraws a sampling of cells.

Lung biopsies can be performed using a variety of techniques. Abronchoscopy is preferably effected to retrieve lung tissues which arelocated deep in the chest. If the area lies close to the chest wall, aneedle biopsy is often done. If both these methods fail, an opensurgical biopsy may be carried out. If there are indications that thelung cancer has spread to the lymph nodes in the mediastinum, amediastinoscopy is performed.

When a needle biopsy is to be done, the subject will be given a sedativeabout an hour before the procedure, to help relaxation. The subject sitsin a chair with arms folded on a table in front. X rays are then takento identify the location of the suspicious areas. Small metal markersare placed on the overlying skin to mark the biopsy site. The skin isthoroughly cleansed with an antiseptic solution, and a local anestheticis injected to numb the area.

A small cut (incision) about half an inch in length is then being made.The subject is asked to take a deep breath and hold it while a specialbiopsy needle is inserted through the incision into the lung. Whenenough tissue has been obtained, the needle is withdrawn. Pressure isapplied at the biopsy site and a sterile bandage is placed over the cut.The entire procedure takes between 30 and 45 minutes.

The subject may feel a brief sharp pain or some pressure as the biopsyneedle is inserted. Most subjects, however, do not experience severepain.

Open biopsies are performed in a hospital under general anesthesia. Aswith needle biopsies, subjects are given sedatives before the procedure.An intravenous line is placed in the arm to give medications or fluidsas necessary. A hollow endotracheal tube, is passed through the throat,into the airway leading to the lungs. It is used to convey the generalanesthetic.

Once the subject is under anesthesia, an incision over the lung area ismade. Some lung tissue is removed and the cut closed with stitches. Theentire procedure takes about an hour. A chest tube is sometimes placedwith one end inside the lung and the other end protruding through theclosed incision. Chest tube placement is done to prevent the lungs fromcollapsing by removing the air from the lungs. The tube is removed a fewdays following the biopsy. A chest X ray is done following an openbiopsy, to check for lung collapse.

The preparation for a mediastinoscopy is similar to that for an openbiopsy. The subject is sedated and prepared for general anesthesia. Theneck and the chest will be cleansed with an antiseptic solution. Oncethe subject is under anesthesia, an incision of about two or threeinches long is made at the base of the neck. A thin, hollow, lightedmediastinoscope is inserted through the cut into the space between theright and the left lungs. The space is examined thoroughly and any lymphnodes or tissues that look abnormal are removed. The mediastinoscope isthen removed, and the incision stitched up and bandaged.

Regardless of the procedure employed, once a biopsy is obtained thelevel of SIM2 can be determined and a diagnosis can thus be made.

Determining a level of SIM2 can be effected using various biochemicaland molecular approaches used in the art for determining geneamplification, and/or level of gene expression.

It will be appreciated that since SIM2 is expressed in normal lung,breast and ovarian tissues at low levels, detection of SIM2 in a normaltissue is preferably effected along side to detect an elevatedexpression and/or amplification. Samples used to determine the normalrange of SIM2 can be normal samples from individuals not suffering fromthe disease condition.

Typically, detection of a nucleic acid of interest in a biologicalsample is effected by hybridization-based assays using anoligonucleotide probe.

The term “oligonucleotide” refers to a single stranded or doublestranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring bases, sugars andcovalent internucleoside linkages (e.g., backbone) as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly to respective naturally-occurring portions. An example of anoligonucleotide probe which can be utilized by the present invention isa single stranded polynucleotide which includes a sequence complementaryto the sequence region encompassed by SEQ ID NO: 9 or 1.

Oligonucleotides designed according to the teachings of the presentinvention can be generated according to any oligonucleotide synthesismethod known in the art such as enzymatic synthesis or solid phasesynthesis. Equipment and reagents for executing solid-phase synthesisare commercially available from, for example, Applied Biosystems. Anyother means for such synthesis may also be employed; the actualsynthesis of the oligonucleotides is well within the capabilities of oneskilled in the art and can be accomplished via established methodologiesas detailed in, for example, “Molecular Cloning: A laboratory Manual”Sambrook et al., (1989); “Current Protocols in Molecular Biology”Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “CurrentProtocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md.(1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley &Sons, New York (1988) and “Oligonucleotide Synthesis” Gait, M. J., ed.(1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramiditefollowed by deprotection, desalting and purification by for example, anautomated trityl-on method or HPLC.

The oligonucleotide of the present invention is of at least 17, at least18, at least 19, at least 20, at least 22, at least 25, at least 30 orat least 40, bases specifically hybridizable with SIM2 derived sequence(e.g., SEQ ID NO: 1 which are hybridizable with the primers set forth inSEQ ID NOs. 4 and 5).

The oligonucleotides of the present invention may comprise heterocylicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′ to 5′ phosphodiester linkage.

Preferably used oligonucleotides are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinunder.

Specific examples of preferred oligonucleotides useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such anoligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. [Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278] and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

It will be appreciated that olignoculeotides of the present inventionmay include further modifications which increase bioavailability,therapeutic efficacy and reduce cytotoxicity. Such modifications aredescribed in Younes (2002) Current Pharmaceutical Design 8:1451-1466.

Hybridization based assays which allow the detection of SIM2 (i.e., DNAor RNA) in a biological sample rely on the use of oligonucleotide whichcan be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to50, more preferably from 40 to 50 nucleotides.

Hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40bp in length) can be effected using the following exampleryhybridization protocols which can be modified according to the desiredstringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI,0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk,hybridization temperature of 1-1.5° C. below the T_(m), final washsolution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH7.6), 0.5% SDS at 1-1.5° C. below the T_(m); (ii) hybridization solutionof 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below theT_(m), final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the T_(m), finalwash solution of 6×SSC, and final wash at 22° C.; (iii) hybridizationsolution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNAand 0.1% nonfat dried milk, hybridization temperature.

The detection of hybrid duplexes can be carried out by a number ofmethods. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Such labels refer to radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. A label can beconjugated to either the oligonucleotide probes or the nucleic acidsderived from the biological sample (target).

For example, oligonucleotides of the present invention can be labeledsubsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, orsome similar means (e.g., photo-cross-linking a psoralen derivative ofbiotin to RNAs), followed by addition of labeled streptavidin (e.g.,phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively,when fluorescently-labeled oligonucleotide probes are used, fluorescein,lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3,Cy3.5, Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others [e.g., Kricka etal. (1992), Academic Press San Diego, Calif.] can be attached to theoligonucleotides.

Traditional hybridization assays include PCR, RT-PCR, RNase protection,in-situ hybridization, primer extension, Southern blot, Northern Blotand dot blot analysis.

Those skilled in the art will appreciate that wash steps may be employedto wash away excess target DNA or probe as well as unbound conjugate.Further, standard heterogeneous assay formats are suitable for detectingthe hybrids using the labels present on the oligonucleotide primers andprobes.

It will be appreciated that a variety of controls may be usefullyemployed to improve accuracy of hybridization assays. For instance,samples may be hybridized to an irrelevant probe and treated with RNAseA prior to hybridization, to assess false hybridization.

Specifically, gene amplification may be measured directly by DNAanalysis such as Southern blot or dot blot techniques. For Southernblotting, DNA is extracted using methods which are well known in theart, involving tissue mincing, cell lysis, protein extraction and DNAprecipitation using 2 to 3 volumes of 100% ethanol, rinsing in 70%ethanol, pelleting, drying and resuspension in water or any othersuitable buffer (e.g., Tris-EDTA). Preferably, following such procedure,DNA concentration is determined such as by measuring the optical density(OD) of the sample at 260 nm (wherein 1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD280 ratio is determined. Preferably, only DNA preparations having an OD260/OD 280 ratio between 1.8 and 2 are used in the following proceduresdescribed hereinbelow.

The purified DNA is then digested with one or more restriction enzymes,and the resulting fragments separated on an agarose gel byelectrophoresis. The DNA fragments are then transferred to a nylon orcellulose nitrate filter by blotting, and the DNA fixed by baking. Thefilter is then exposed to a labeled complementary probe and the regionsof hybridization detected, usually by autoradiography. Dot blotting issimilar, except that the DNA fragments are not separated on the gel. Thedegree of gene amplification is then determined by dilutional analysisor densitometry scanning.

Polymerase chain reaction (PCR)-based methods may be used to identifythe presence of SIM2 mRNA. For PCR-based methods a pair ofoligonucleotides is used, which is specifically hybridizable with theSIM2 polynucleotide sequences described hereinabove in an oppositeorientation so as to direct exponential amplification of a portionthereof (including the hereinabove described sequence alteration) in anucleic acid amplification reaction. For example, an oligonucleotidepair of primers specifically hybridizable with SIM2 are set forth in SEQID NOs: 10 and 11 which provide an amplification product whichcorresponds to SEQ ID NO: 9.

The polymerase chain reaction and other nucleic acid amplificationreactions are well known in the art and require no further descriptionherein. The pair of oligonucleotides according to this aspect of thepresent invention are preferably selected to have compatible meltingtemperatures (Tm), e.g., melting temperatures which differ by less thanthat 7° C., preferably less than 5° C., more preferably less than 4° C.,most preferably less than 3° C., ideally between 3° C. and 0° C.

Hybridization to oligonucleotide arrays may be also used to determineSIM2 expression. Such screening has been undertaken in the BRCA1 geneand in the protease gene of HIV-1 virus [see Hacia et al., (1996) NatGenet 1996; 14(4):441-447; Shoemaker et al., (1996) Nat Genet 1996;14(4):450-456; Kozal et al., (1996) Nat Med 1996; 2(7):753-759].

The nucleic acid sample which includes the candidate region to beanalyzed is isolated, amplified and labeled with a reporter group. Thisreporter group can be a fluorescent group such as phycoerythrin. Thelabeled nucleic acid is then incubated with the probes immobilized onthe chip using a fluidics station. For example, Manz et al. (1993) Advin Chromatogr 1993; 33:1-66 describe the fabrication of fluidics devicesand particularly microcapillary devices, in silicon and glasssubstrates.

Once the reaction is completed, the chip is inserted into a scanner andpatterns of hybridization are detected. The hybridization data iscollected, as a signal emitted from the reporter groups alreadyincorporated into the nucleic acid, which is now bound to the probesattached to the chip. Since the sequence and position of each probeimmobilized on the chip is known, the identity of the nucleic acidhybridized to a given probe can be determined.

It will be appreciated that when utilized along with automatedequipment, the above described detection methods can be used to screenmultiple samples for ovarian, breast or lung cancers both rapidly andeasily.

The presence of SIM2 in an ovarian, breast and/or lung tissue can alsobe determined at the protein level. Numerous protein detection assaysare known in the art, examples include but are not limited tochromatography, electrophoresis, immunodetection assays such as ELISAand western blot analysis, immunohistochemistry and the like, which maybe effected using antibodies specific to SIM2. Thus, the presentinvention envisages the use of serum immunoglobulins, polyclonalantibodies or fragments thereof, (i.e., immunoreactive derivativesthereof), or monoclonal antibodies or fragments thereof for thedetection of ovarian, breast and/or lung cancer. Monoclonal antibodiesor purified fragments of the monoclonal antibodies having at least aportion of an antigen-binding region, including the fragments describedhereinbelow, chimeric or humanized antibodies and complementarilydetermining regions (CDR).

The term “antibody” refers to whole antibody molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, and Fv that arecapable of binding with antigenic portions of the target polypeptide.These functional antibody fragments constitute preferred embodiments ofthe present invention, and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule as described in, for example,U.S. Pat. No. 4,946,778.

SIM2-specific antibodies can be commercially obtained from Santa Cruz[SIM21 (C-17):sc-8716; SIM2s (C-15):sc-8715].

Alternatively, SIM2 specific antibodies may be generated using methods,which are well known in the art. See for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork, 1988, incorporated herein by reference).

Purification of serum immunoglobulin antibodies (polyclonal antisera) orreactive portions thereof can be accomplished by a variety of methodsknown to those of skill including, precipitation by ammonium sulfate orsodium sulfate followed by dialysis against saline, ion exchangechromatography, affinity or immunoaffinity chromatography as well as gelfiltration, zone electrophoresis, etc. (see Goding in, MonoclonalAntibodies: Principles and Practice, 2nd ed., pp. 104-126, 1986,Orlando, Fla., Academic Press). Under normal physiological conditionsantibodies are found in plasma and other body fluids and in the membraneof certain cells and are produced by lymphocytes of the type denoted Bcells or their functional equivalent. Antibodies of the IgG class aremade up of four polypeptide chains linked together by disulfide bonds.The four chains of intact IgG molecules are two identical heavy chainsreferred to as H-chains and two identical light chains referred to asL-chains. Additional classes include IgD, IgE, IgA, IgM and relatedproteins.

Methods of generating and isolating monoclonal antibodies are well knownin the art, as summarized for example in reviews such as Tramontano andSchloeder, Methods in Enzymology 178, 551-568, 1989. A recombinant SIM2polypeptide may be used to generate antibodies in vitro. Morepreferably, the recombinant is used to elicit antibodies in vivo. Ingeneral, a suitable host animal is immunized with the recombinant SIM2.Advantageously, the animal host used is a mouse of an inbred strain.Animals are typically immunized with a mixture comprising a solution ofthe recombinant SIM2 in a physiologically acceptable vehicle, and anysuitable adjuvant, which achieves an enhanced immune response to theimmunogen. By way of example, the primary immunization conveniently maybe accomplished with a mixture of a solution of the recombinant SIM2 andFreund's complete adjuvant, said mixture being prepared in the form of awater in oil emulsion. Typically the immunization will be administeredto the animals intramuscularly, intradermally, subcutaneously,intraperitoneally, into the footpads, or by any appropriate route ofadministration. The immunization schedule of the immunogen may beadapted as required, but customarily involves several subsequent orsecondary immunizations using a milder adjuvant such as Freund'sincomplete adjuvant. Antibody titers and specificity of binding to theSIM2 can be determined during the immunization schedule by anyconvenient method including by way of example radioimmunoassay, orenzyme linked immunosorbant assay, which is known as the ELISA assay.When suitable antibody titers are achieved, antibody-producinglymphocytes from the immunized animals are obtained, and these arecultured, selected and cloned, as is known in the art. Typically,lymphocytes may be obtained in large numbers from the spleens ofimmunized animals, but they may also be retrieved from the circulation,the lymph nodes or other lymphoid organs. Lymphocytes are then fusedwith any suitable myeloma cell line, to yield hybridomas, as is wellknown in the art. Alternatively, lymphocytes may also be stimulated togrow in culture, and may be immortalized by methods known in the artincluding the exposure of these lymphocytes to a virus, a chemical or anucleic acid such as an oncogene, according to established protocols.After fusion, the hybridomas are cultured under suitable cultureconditions, for example in multi-well plates, and the culturesupernatants are screened to identify cultures containing antibodiesthat recognize the hapten of choice. Hybridomas that secrete antibodiesthat recognize the recombinant SIM2 are cloned by limiting dilution andexpanded, under appropriate culture conditions. Monoclonal antibodiesare purified and characterized in terms of immunoglobulin type andbinding affinity.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety (see also Porter, R. R., Biochem. J., 73: 119-126, 1959).Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al. (Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972). Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, all of which are hereby incorporated, byreference, in entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick and FryMethods, 2: 106-10, 1991).

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues, which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source, which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human monoclonal antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Once the level of SIM2 is determined and the subject diagnosed, thediagnosis can be further validated using other diagnostic methods, whichmay also provide an accurate staging of the disease in the case of apositive diagnosis. Thus, for example, positive diagnosis for ovariancancer may be confirmed by transabdominal/transvaginal ultrasonographyand/or a blood test for the tumor marker CA-125. Ultrasound screeninginvolves looking for enlarged ovaries and a transvaginal colour Dopplerultrasound is used to image blood flow. Blood vessel formation isthought to discriminate between cancer and benign cysts. Alternatively,positive diagnosis of lung cancer using the method of the presentinvention can be validated using chest X-rays and sputum cytology aswell as spiral computed tomography (CT) scanning which can detect evenvery small tumors. Positive diagnosis of breast cancer using the methodof the present invention can be validated using scintimammography,mammography, ultrasound and/or magnetic resonance imaging (MRI).

It will be appreciated that the above-described method of this aspect ofthe present invention may also be used to monitor disease progressionand therapeutic regimen.

In addition to diagnostic advances pioneered by the present invention,the identification of overexpression of SIM2 in ovarian and lung cancersallows for the design of therapeutic agents, which can be used to treatovarian, breast and lung cancers.

Thus, according to another aspect of the present invention there isprovided a method of treating a subject (i.e., mammal e.g., human)having ovarian cancer, breast and/or lung cancer.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of ovarian cancer and/or lung cancer.

The method according to this aspect of the present invention is effectedby specifically downregulating expression or activity of SIM2 in a lungtissue, breast tissue and/or ovarian tissue to thereby treat the ovariancancer, breast cancer and/or lung cancer in the subject.

Preferably, the method is effected by providing to the subject atherapeutically effective amount of an agent which is capable ofdownregulating SIM2 expression and/or activity.

As used herein “an agent capable of downregulating SIM2 expressionand/or activity” refers to a molecule, which is capable of directly orindirectly down-regulating SIM2 expression or activity. An agent fordirect downregulation of SIM2 refers to a molecule, which inhibits SIM2intrinsic activity or expression. An agent for indirect downregulationof SIM2 refers to a molecule which inhibits the activity of a S[M2effector (e.g., ARNT and HIF-1α) or expression thereof. The agentsaccording to this aspect of the present invention can be a moleculewhich binds SIM2 (e.g., an antibody); an enzyme which cleaves SIM2; anantisense polynucleotide capable of specifically hybridizing with anmRNA transcript encoding SIM2; a SIM2 specific aptamer, a ribozyme whichspecifically cleaves SIM2 transcripts; a non-functional analogue of atleast a catalytic or binding portion of SIM2 (e.g., a peptide-agentwhich can be identified using a phage display technology); a moleculewhich prevents SIM2 activation or substrate binding; an siRNA moleculecapable of inducing degradation of SIM2 transcripts; a DNAzyme whichspecifically cleaves SIM2 transcripts or DNA, an activateddouble-stranded RNA (dsRNA-dependent protein kinase PKR as described in[Shir and Levitzki (2002) Nat. Biotechnol. 20(9):895-900 and Cell MolNeurobiol. (2001) 21(6):645-56] and a molecule which is capable ofpromoting SIM2 specific immunization response.

One example, of an agent capable of downregulating a SIM2 is an antibodyor antibody fragment capable of specifically binding SIM2 or an effectorthereof. Examples of SIM2 antibodies are described hereinabove.Preferably, such antibodies are directed at functional domains of atarget protein. Thus, for example, a SIM2 antibody according to thisaspect of the present invention is preferably directed at the effectorbinding domain such as the ARNT binding domain. Alternatively, theantibody may bind a SIM2 effector such as ARNT or HIF-1α. An anti ARNTpolyclonal rabbit serum raised against residues 1-140 of human ARNT andan anti HIF-1α polyclonal serum raised against residues 786-826 of humanHIF-1α have been described by Susan (2002) J. Biol. Chem.277:10236-10243. Preferably, the antibodies, according to this aspect ofthe present invention, are humanized such as described hereinabove.

Alternatively an agent capable of downregulating a SIM2 or an effectorthereof can be a protease, which is designed to cleave SIM2. Proteaseswhich can be used to cleave SIM2 can be identified by performing acomputational analysis such as by using the SMART, MEME, MOTIFS,CDD-NCBI, BLOCKS or mPredict software each available fromhttp://molbio.info.nih.gov/talks/tools/jobs.html and identifying aprotease cleavage site.

Alternatively, agents which are designed to inhibit functional domainsin the SIM2 protein (i.e., protein-protein interaction domains) can becomputationally identified. For example, various peptide sequencesderived from SIM2 can be computationally analyzed for an ability to bindan inhibitor using a variety of three dimensional computational tools.Software programs useful for displaying three-dimensional structuralmodels, such as RIBBONS (Carson, M., 1997. Methods in Enzymology 277,25), 0 (Jones, T A. et al., 1991. Acta Crystallogr. A47, 110), DINO(DINO: Visualizing Structural Biology (2001) http://www.dino3d.org); andQUANTA, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP(reviewed in Kraulis, J., 1991. Appl Crystallogr. 24, 946) can beutilized to model interactions between SIM2 and prospective peptideand/or other small molecule inhibitors. Computational modeling ofprotein-peptide interactions has been successfully used in rational drugdesign, for further detail, see Lam et al., 1994. Science 263, 380;Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993.Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993.Perspectives in Drug Discovery and Design 1, 109, and Mauro M J. et al.,2002. J Clin Oncol. 20, 325-34. Specifically, the PRO_SELECT, tool forthe virtual screening of libraries for fit to a protein active site, hasbeen used to find novel leads against the serine protease factor Xa[Liebeschuetz J Med Chem. (2002);45(6):1221-32].

Another agent capable of downregulating a SIM2 or an effector thereof isa small interfering RNA (siRNA) molecule. RNA interference is a two-stepprocess. the first step, which is termed as the initiation step, inputdsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs(siRNA), probably by the action of Dicer, a member of the RNase IIIfamily of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA(introduced directly or via a transgene or a virus) in an ATP-dependentmanner. Successive cleavage events degrade the RNA to 19-21 bp duplexes(siRNA), each with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex tofrom the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the SIM2 mRNA sequence of interest(e.g., SEQ ID NO: 7) is scanned downstream of the AUG start codon for AAdinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19nucleotides is recorded as potential siRNA target sites. Preferably,siRNA target sites are selected from the open reading frame, asuntranslated regions (UTRs) are richer in regulatory protein bindingsites. UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNA endonuclease complex [TuschlChemBiochem. 2:239-245]. It will be appreciated though, that siRNAsdirected at untranslated regions may also be effective, as demonstratedfor GAPDH wherein siRNA directed at the 5′ UTR mediated about 90%decrease in cellular GAPDH mRNA and completely abolished protein level(www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. siRNA target sites on SEQ ID NO: 1 are preferably selectedfrom the 5′ end i.e., coordinates 1-304 (see FIG. 1) e.g., nucleotidecoordinates 171-193 of SEQ ID NO: 1. For better evaluation of theselected siRNAs, a negative control is preferably used in conjunction.Negative control siRNA preferably include the same nucleotidecomposition as the siRNAs but lack significant homology to the genome.Thus, a scrambled nucleotide sequence of the siRNA is preferably used,provided it does not display any significant homology to any other gene.

Another agent capable of downregulating a SIM2 or an effector thereof isa DNAzyme molecule capable of specifically cleaving an mRNA transcriptor DNA sequence of the SIM2. DNAzymes are single-strandedpolynucleotides which are capable of cleaving both single and doublestranded target sequences (Breaker, R. R. and Joyce, G. Chemistry andBiology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad.Sci. USA 1997; 943:4262) A general model (the “10-23” model) for theDNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of15 deoxyribonucleotides (e.g., nucleotide coordinates 311-326 of SEQ IDNO: 1), flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions (Santoro, S. W. &Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes seeKhachigian, LM [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al,20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). Inanother application, DNAzymes complementary to bcr-abl oncogenes weresuccessful in inhibiting the oncogenes expression in leukemia cells, andlessening relapse rates in autologous bone marrow transplant in cases ofCML and ALL.

Downregulation of a SIM2 or an effector thereof can also be effected byusing an antisense polynucleotide capable of specifically hybridizingwith an mRNA transcript encoding the SIM2 transcripts.

Design of antisense molecules, which can be used to efficientlydownregulate a SIM2 must be effected while considering two aspectsimportant to the antisense approach. The first aspect is delivery of theoligonucleotide into the cytoplasm of the appropriate cells, while thesecond aspect is design of an oligonucleotide which specifically bindsthe designated mRNA within cells in a way which inhibits translationthereof. An example antisense oligonucleotide which can be used inaccordance with the present invention is designed to hybridize tonucleotide coordinates 311-400 of SEQ ID NO: 2. Such an antisensemolecule hybridizes to a sequence which is shared by all SIM2 expressionproducts known to date and as such may be useful in silencing SIM2expression.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997);and Aoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60(2001)].

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

It will be appreciated that antisense oligonucleotides can be used tomodulate alternative splicing from SIM2 gene [Suzani and Kole (2003)Progress in Molecular and Subcellular Biology vol. 31 Philippe Jeanteur(Ed.) Springer-Verlag Berlin Heidelberg]. Inhibition of splicing byantisense oligonucleotides can be accomplished by targetingoligonucleotides to small nuclear RNAs (snRNAs), which participate inspliceosome formation and are essential for splicing and to splice sitesand adjacent sequences [reviewed in Kole (1991) Adv. Drug Delivery Rev.6:271-286].

Another agent capable of downregulating a SIM2 or an effector thereof isa ribozyme molecule capable of specifically cleaving an mRNA transcriptencoding a SIM2 for example. Ribozymes are being increasingly used forthe sequence-specific inhibition of gene expression by the cleavage ofmRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol.9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)].Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms have demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays (RibozymePharmaceuticals, Incorporated—WEB home page).

Alternatively, the agent can be a molecule, which promotes aSIM2-specific immunogenic response in the subject. The molecule can be aSIM2 protein, a fragment derived therefrom or a nucleic acid sequenceencoding thereof. Although such a molecule can be provided to thesubject per se, the agent is preferably administered with animmunostimulant in an immunogenic composiiton. An immunostimulant may beany substance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes into which the compound isincorporated (see e.g., U.S. Pat. No. 4,235,877). Vaccine preparation isgenerally described in, for example, M. F. Powell and M. J. Newman,eds., “Vaccine Design (the subunit and adjuvant approach),” Plenum Press(NY, 1995).

Illustrative immunogenic compositions may contain DNA encoding one ormore of the SIM2 polypeptides as described above, such that thepolypeptide is generated in situ. The DNA may be present within any of avariety of delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems (see below), bacteria andviral expression systems. Numerous gene delivery techniques are wellknown in the art, such as those described by Rolland, Crit. Rev. Therap.Drug Carrier Systems 15:143-198, 1998, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the subject (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope. In a preferred embodiment, the DNA may beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

It will be appreciated that an immunogenic composition may comprise botha polynucleotide and a polypeptide component. Such immunogeniccompositions may provide for an enhanced immune response.

Any of a variety of immunostimulants may be employed in the immunogeniccompositions of this invention. For example, an adjuvant may beincluded. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2,-7, or -12, may alsobe used as adjuvants.

The adjuvant composition may be designed to induce an immune responsepredominantly of the Th1 type. High levels of Th1-type cytokines (e.g.,IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the induction ofcell mediated immune responses to an administered antigen. In contrast,high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10)tend to favor the induction of humoral immune responses. Followingapplication of an immunogenic composition as provided herein, thesubject will support an immune response that includes Th1- and Th2-typeresponses. The levels of these cytokines may be readily assessed usingstandard assays. For a review of the families of cytokines, see Mosmannand Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available fromSmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720.

A delivery vehicle may be employed within the immunogenic composition ofthe present invention to facilitate production of an antigen-specificimmune response that targets tumor cells. Delivery vehicles includeantigen presenting cells (APCs), such as dendritic cells, macrophages, Bcells, monocytes and other cells that may be engineered to be efficientAPCs. Such cells may be genetically modified to increase the capacityfor presenting the antigen, to improve activation and/or maintenance ofthe T cell response, to have anti-tumor effects per se and/or to beimmunologically compatible with the receiver (i.e., matched HLAhaplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmernan and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within an immunogenic composition (seeZitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNF.alpha. to cultures of monocytes harvestedfrom peripheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligandand/or other compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are categorized as “immature” and “mature” cells, whichallows a simple way to discriminate between two well characterizedphenotypes. Immature dendritic cells are characterized as APC with ahigh capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding SIM2,such that SIM2, or an immunogenic portion thereof, is expressed on thecell surface. Such transfection may take place ex vivo, and acomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to the subject, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the SIM2 polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule) such as described above. Alternatively, a dendriticcell may be pulsed with a non-conjugated immunological partner,separately or in the presence of the polypeptide.

Agents for downregulating expression or activity of SIM2 (i.e., activeingredients) of the present invention can be provided to the subject perse, or as part of a pharmaceutical composition where they are mixed witha pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carbomethylcellulose; and/or physiologically acceptable polymerssuch as polyvinylpyrrolidone (PVP). If desired, disintegrating agentsmay be added, such as cross-linked polyvinyl pyrrolidone, agar, oralginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Pharmaceutical compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert.

It will be appreciated that the oligonucleotides of the presentinvention can also be expressed from a nucleic acid construct, which canbe administered to the subject employing any suitable mode ofadministration, described hereinabove (e.g., in-vivo gene therapy). Sucha nucleic acid construct is introduced into a target cell or cells viaappropriate gene delivery vehicle/methods (transfection, transduction,homologous recombination, etc.) and an expression system as needed andthen the modified cells are expanded in culture and returned to thesubject (i.e., ex-vivo gene therapy).

Such expression constructs may include a tissue-specific promoter fordirecting expression of the downregulating agents in the malignanttissue. Thus an ovarian specific promoter such as OSP-1 [Kumaran CancerRes. (2001) February 15; 61(4):1291-5] and IAI.3B [Hamada Cancer Res.(2003) May 15; 63(10):2506-12] may be used. A breast-specific promoterwhich may be used in accordance with the present invention includesrNRL, Muc-1 and mWAP [Hiripi (2003) DNA Cell Biol. 22:41-5; Berger(2001) Breast Cancer Res. 3:28-35]. Alternatively a lung specificpromoter such as CC-10 [Harrod Am J Respir Cell Mol. Biol. (2002)February; 26(2):216-23]and SP-C [Duan Oncogene. (2002) November 7;21(51):7831-8]may be used Expression of duplex oligonucleotides ispreferably effected via expression vectors specifically designed forsuch use. For example, the pSUPER™ including the polymerase-III H1-RNAgene promoter with a well defined start of transcription and atermination signal consisting of five thymidines in a row (T5)[Brummelkamp (2002) Science 296:550-53]. Another suitable siRNAexpression vector encodes the sense and antisense siRNA under theregulation of separate polIII promoters [Miyagishi and Taira[(2002)Nature Biotech. 20:497-500]. The resultant siRNA includes 5 thymidinetermination signal. Alternatively, oligonucleotide sequences can beplaced under bi-directional promoters to produce both the sense andantisense transcripts from the same promoter construct, thus simplifyingthe construction of expression vectors and achieving an equal molarratio of cellular sense and antisense sequences. Examples forbi-directional promoters are disclosed in U.S. Pat. Appl. No.20020108142. It will be appreciated that when duplex oligonucleotide areused, transfection reagents dedicated to siRNA transfer to mammaliancells are preferably employed. Examples for such include but are notlimited to siPORT™ Amine (i.e., a polyamine mixture) and siPORT™ Lipid(i.e., a mixture of cationic and neutral lipids).

Accordingly, in cases where the duplex oligonucleotides of the presentinvention are introduced into a cell in which RNA interference (RNAi)does not normally occur, the factors needed to mediate RNAi areintroduced into such a cell or the expression of the needed factors isinduced, as disclosed in U.S. Pat. Appl. No.: 20020086356.

It will be appreciated that treatment of subjects exhibiting mutatedSIM2 transcripts may also be effected using a “knock in” strategy (seeU.S. Pat. No. 6,265,632), wherein endogenous SIM2 sequence alterationsare corrected using advanced gene therapy.

As is mentioned hereinabove, the present inventors uncovered novelisoforms of SIM-2.

Thus, according to another aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding a polypeptide being at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 82%, at least 86%, at least 88%, atleast 90%, at least 92%, at least 94% or more, say 95%-100% homologousto SEQ ID NO: 39, as determined using the BestFit software of theWisconsin sequence analysis package, utilizing the Smith and Watermanalgorithm, where the gap creation equals 8 and gap extension penaltyequals 2.

According to one preferred embodiment of this aspect of the presentinvention the isolated polynucleotide is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 82%, at least 86%, atleast 88%, at least 90%, at least 92%, at least 94% or more, say95%-100% identical to SEQ ID NO: 39, as determined using BestFitsoftware of the Wisconsin sequence analysis package, utilizing the Smithand Waterman algorithm, where gap weight equals 50, length weight equals3, average match equals 10 and average mismatch equals −9.

According to another preferred embodiment of this aspect of the presentinvention the isolated polynucleotide is as set forth in SEQ ID NO: 2.

According to yet another aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding a polypeptide being at least 50%, at least 5%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 82%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94% ormore, say 95%-100% homologous to SEQ ID NOs: 40 or 41, as determinedusing the BestFit software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where the gap creationequals 8 and gap extension penalty equals 2.

According to one preferred embodiment of this aspect of the presentinvention the isolated polynucleotide is at least 50%, at least 5%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 82%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94% or more, say 95%-100% identical to SEQ ID NOs: 40 or 41, asdetermined using BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap weightequals 50, length weight equals 3, average match equals 10 and averagemismatch equals −9.

According to another preferred embodiment of this aspect of the presentinvention the isolated polynucleotide is as set forth in SEQ ID NO: 3.

As used herein the phrase “an isolated polynucleotide” refers to asingle or double stranded nucleic acid sequences which is isolated andprovided in the form of an RNA sequence, a complementary polynucleotidesequence (cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

Since the polynucleotide sequences of the present invention encodepreviously unidentified polypeptides, the present invention alsoencompasses isolated polypeptides or portions thereof which are encodedby the isolated polynucleotide which are described hereinabove.

Thus, this aspect of the present invention also encompasses polypeptideswhich are set forth in SEQ ID NO: 39, 40 or 41, homologues thereof(selected from the homology range of 60-100% described hereinabove)fragments thereof and altered polypeptides characterized by mutations,such as deletion, insertion or substitution of one or more amino acids,either naturally occurring or man induced, either randomly or in atargeted fashion.

Since expression of SIM2 is correlatable with cancer development (see WO02/12565) the present invention also envisages the use of the novelsequences in diagnosis and treatment of cancer. Examples include, butare not limited to bone cancers, brain tumors, breast cancer, endocrinesystem cancers, gastrointestinal cancers, gynecological cancers, headand neck cancers, leukemia, lymphomas, metastases, myelomas, pediatriccancers, penile cancer, prostate cancer, sarcomas, skin cancers, thyroidcancer, thyoma, urinary tract cancers, carcinoma of unknown primary andLi-Fraumeni syndrome.

As is illustrated in the Examples section which follows, the presentinventors have shown through laborious experimentation that thesesequences are differentially expressed in colon adenocarcinoma, in lungadenocarcinoma and in lung squamous cell carcinoma supporting the use ofsuch sequences in diagnosis and treatment of cancer as described above.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes 1-111 Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Genomic Organization of SIM2

Schematic presentation of SIM2 lung specific transcripts is shown inFIG. 1. Specifically, the genomic alignment of the exons of SEQ ID NOs:1-3, 7 and 8 is shown. The genomic sequence used as a reference ischromosome 21q22.13, starting at position 36992386 and terminating atposition 37042613. Genomic sequences are available at UCSC GenomeBioinformatics database, release version April 2002(http:/genome.ucsc.edu).

Table 1, below shows the coordinates of the exons on the genomicsequence for each SEQ ID NO, correspondingly. TABLE 1 Relative exon Exoncoordinates on coordinates on SEQ ID NO: expressed sequence genomicsequence 1 1-1001 12073-13073 2 1-312 12073-12384 2 313-404 13843-139322 405-513 21132-21240 2 514-599 24356-24441 2 600-621 27430-27451 31-517 1001-1517 3 518-1766 1930-3178 3 1767-3143 5613-6989 3 3144-34737432-7761 3 3474-3640 8343-8509 3 3541-3895 9165-9419 3 3896-41599589-9852 3 4160-4644 10281-10765 3 4645-8296 11543-15194 7 1-2311001-1231 7 232-314 10478-10560 7 315-404 13843-13932 7 405-51321132-21240 7 514-599 24356-24441 7 600-799 27430-27629 7 800-90632356-32462 7 907-1054 43028-43175 7 1055-1223 44698-44866 7 1224-163246039-46447 7 1633-3885 48976-51228 8 1-231 1001-1231 8 232-31410478-10560 8 315-404 13843-13932 8 405-513 21132-21240 8 514-59924356-24441 8 600-799 27430-27629 8 800-906 32356-32462 8 907-105443028-43175 8 1055-1223 44698-44866 8 1224-2823 46039-46447

Example 2 Materials and Exprerimental Procedures

RNA preparation—RNA was commercially obtained from Clontech (FranklinLakes, N.J. USA 07417, www.clontech.com) or BioChain Inst. Inc.(www.biochain.com) or ABS or Clinomics. Alternatively RNA was purifiedfrom tissue samples using TR1-Reagent (Molecular Research Center),according to Manufacturer's instructions. Tissue samples were obtainedfrom subjects or from postmortem. Total RNA samples were treated withDNaseI (Ambion) then purified using RNeasy columns (Qiagen).

Lung—Lu_(—)1N-Lu_(—)5N sample was prepared from commercially availablenormal lung RNAs (BioChain, Cat. No. CDP-061010, Lot Nos: A503205,A503384, A503385, A503204, A503206). RT Lu_(—)6N was prepared from apool of 6 normal lung RNAs (BioChain, Cat. No. CDP-061010, Lot No.A409363).

Lu_(—)7C— Lu_(—)16C samples were prepared from lung adenocarcinoma RNAs:samples Lu_(—)7C— Lu_(—)10C were prepared from commercially availableRNAs (BioChain, Cat. No. CDP-064004A, Lot Nos: A504117, A504119,A504116, A504118), and samples Lu_(—)11C-Lu_(—)16C were prepared fromRNA purified from tissue samples of subjects.

RTs Lu_(—)17C-Lu_(—)31C were prepared from squamous cell carcinoma RNAs:samples Lu_(—)17C-Lu_(—)22C were prepared from commercial RNAs(BioChain, Cat. No. CDP-064004B, Lot Nos: A503187, A503386, A503387,A503183, A411075), sample Lu_(—)27C was prepared from commercial RNA(Clontech, Cat No: 64013-1), samples Lu_(—)28C-Lu_(—)30C were preparedfrom commercial RNAs (BioChain, Cat. No. CDP-064004, Lot Nos: A409017,A409091, A408175), and samples Lu_(—)23C-Lu_(—)26C and Lu_(—)31C wereprepared from RNA purified from subjects tissue samples.

Lu_(—)32C-Lu_(—)35C were prepared from commercial small cell carcinomaRNAs (BioChain, Cat. No. CDP-064004D, Lot Nos: A504115, A501390,A501389, A501391).

Lu_(—)36C-Lu_(—)37C were prepared from commercial large cell carcinomaRNAs (BioChain, Cat. No. CDP-064004C, Lot Nos: A504113, A504114).

Lu_(—)38C was prepared from commercial alveolus cell carcinoma RNAs(BioChain, Cat. No. CDP-064004, Lot Nos: A409089).

RT Lu_(—)39C was prepared from lung carcinoma RNA purified from subjecttissue sample (with no forther subcaracterization).

Lu_(—)40H1299 was prepared from RNA purified from NCI_H1299 cell linenon-small cell carcinoma (ATCC Catalog No: CRL-5803).

SG_(—)41 was prepared from commercial normal salivary gland RNA (pool of24) (Clontech, Cat No: 64110-1).

Lu_(—)16N and Lu_(—)25N were prepared from RNA purified from subjectsnormal tissue samples matched to the cancer samples Lu_(—)16C andLu_(—)25C.

Colon—RTs marked as “Col_xN” (Col_(—)2N-Col_(—)276N) were prepared fromnormal colon RNAs. Samples Col_(—)22N and Col_(—)23N were prepared fromcommercial RNAs (BioChain, Cat. No. CDP-064007, Lot Nos: A501132,A501130), and the rest were prepared from RNA purified from normalsubjects tissue samples. Normal pool sample was prepared form comercialRNA pool of normal colon (BioChain, Cat. No. CDP-061003, Lot Nos:A411078).

RTs Col_(—)2C-Col_(—)23C were prepared from colon adenocarcinoma RNAs,matched to the normal samples Col_(—)2N-Col_(—)23N. Samples Col_(—)22Cand Col_(—)23C were prepared from commercial RNAs BioChain, Cat. No.CDP-064007, Lot Nos: A501131, A501129), and the rest were prepared fromRNA purified from subjects colon adenocarcinoma cancer tissue samples.

Colon cell lines—Col-SW620 is epithelial colorectal adenocarcinoma frommetastatic lymph node, Dukes C (ATCC Catalog No: CCL-227), Col-SW480 isepithelial colorectal adenocarcinoma, Dukes B (ATCC Catalog No: CCL-228)and Col-DLD1 is epithelial colorectal adenocarcinoma, Dukes C (ATCCCatalog No: CCL-221).

Ovary—Ovarian RNA was generated as described in Table 2, below. TABLE 2Serial number Lot number Source Tissue Pathology (grade) 1-Pap Adeno G3ILS-1406 ABS ovary papillary adenocarcinoma (3) 2-Pap Adeno G2 ILS-1408ABS ovary papillary adenocarcinoma (2) 3-Pap Adeno G2 ILS-1431 ABS ovarypapillary adenocarcinoma (2) 4-Pap CystAde G2 ILS-7286 ABS ovarypapillary cystadenocarcinoma (2) 5-Adeno G3 99-12-G432 GOG ovaryadenocarcinoma (3) 6-Adeno G3 A0106 ABS ovary adenocarcinoma (3) 7-AdenoG3 IND-00375 ABS ovary adenocarcinoma (3) 8-Adeno G3 A501113 BioChainovary adenocarcinoma (3) 9-Adeno G3 99-06-G901 GOG ovary adenocarcinoma(maybe serous) (3) 10-Adeno G3 A407069 Biochain ovary adenocarcinoma (3)11-Adeno G3 A407068 Biochain ovary adenocarcinoma (3) 12-Adeno G3A406023 Biochain ovary adenocarcinoma (3) 13-Adeno G3 94-05-7603 GOGright ovary metastasis adenocarcinoma (3) 14-Adeno G2 A501111 BioChainovary adenocarcinoma (2) 15-Carcinoma G3 A407065 BioChain ovarycarcinoma (3) 16-Carcinoma 1090387 Clontech ovary carcinoma NOS 17-MucAdeno G3 A504084 BioChain ovary mucinous adenocarcinoma (3) 18-Muc AdenoG3 A504083 BioChain ovary mucinous adenocarcinoma (3) 19-Muc Adeno G3A504085 BioChain ovary mucinous adenocarcinoma 20-Pap Muc CystAdeUSA-00273 ABS ovary papillary mucinous cystadenocarcinoma 21-Muc CystAdeG2-3 95-10-G020 GOG ovary mucinous cystadenocarcinoma (2- 3) 22-MucCystAde G2 A0139 ABS ovary mucinous cystadenocarcinoma (2) 23-MucCystAde G3 VNM- ABS ovary mucinous cystadenocarcinoma 00187 with lowmalignant (3) 24-Pap Sero Adeno G3 2001-07- GOG ovary papillary serousadenocarcinoma G801 (3) 25-Pap Sero Adeno G3C N0021 ABS ovary papillaryserous adenocarcinoma (3C) 26-Sero Adeno G3 2001-12- GOG right ovaryserous adenocarcinoma (3) G035 27-Pap Sero Carci G3 2001-08- GOG ovarypapillary serous carcinoma (3) G011 28-Pap Sero CystAde G3 A503176BioChain ovary serous papillary cystadenocarcinoma (3) 29-Pap SeroCystAde G3 93-09-4901 GOG ovary serous papillary cystadenocarcinoma (3)30-Pap Sero CystAde G3 A503175 BioChain ovary serous papillarycystadenocarcinoma (1) 31-Pap Endo Adeno G3C 95-04-2002 GOG ovarypapillary endometrioid adenocarcinoma (3C) 32-Endo Adeno G2 94-08-7604GOG right ovary endometrioid adenocarcinoma (2) 33-Endo Adeno G1-22000-09- GOG ovary endometrial adenocarcinoma (1-2) G621 34-MixSero/Endo G3 2002-05- GOG ovary mixed serous and endometrioid G513adenocarcinoma (3) 35-Mix Sero/Endo G3 2002-05- GOG ovary mixed serousand endometrioid G509 adenocarcinoma of mullerian (3) 36-Mix Sero/EndoG3 2001-12- GOG ovary mixed serous and endometrioid G037 adenocarcinoma(3) 37-Mix Sero/Endo G2 95-11-G006 GOG ovary, papillary serous andendometrioid endometrium cystadenocarcinoma (2) 38-Mix Sero/Muc/Endo G298-03-G803 GOG ovary mixed epithelial cystadenocarcinoma with mucinous,endometrioid, squamous and papillary serous(2) 39-Adeno borderline98-08-G001 GOG ovary epithelial adenocarcinoma of borderline malignancy40-Clear cell Adeno G3 2001-10- GOG ovary clear cell adenocarcinoma (3)G002 41-Clear cell Adeno 2001-07- GOG ovary clear cell adenocarcinomaG084 42-N A503274 BioChain ovary Normal 43-N A504086 BioChain ovaryNormal 44-N 061P43A Ambion ovary Normal 45-N A504087 BioChain ovaryNormal 46-N M14 A501112 BioChain ovary Normal (matched tumor A501111)47-N M8 A501114 BioChain ovary Normal (matched tumor A501113) 48-N M3898-03- GOG ovary Normal (matched tumor 98-03- G803N G803) 49-N M3998-08- GOG ovary Normal (matched tumor 98-08- G001N G001)

RT reaction with oligo-dT—Reverse transcription was effected using 2 μgof total RNA, in a 20 μl reaction, including 200 units of Superscript IIReverse Transcriptase (Bibco/BRL) in the buffer supplied by themanufacturer, 500 pmol of oligo(dT)₂₅ (Promega Corp. Madison Wis., USA),and 40 units of RNasin (Promega Corp. Madison Wis., USA).

Real Time PCR—5 ul RT reaction products, diluted in final reactionvolume of 20 ul was used for amplification. Specific oligonucleotides(SEQ ID NO:4 and SEQ ID NO:5) were used as primers. The ABI Prism 7000Sequence Detection System was used for cycling. The reaction waseffected using SYBR GreenPCR Master Mix (Applied Biosystems). The cyclein which the reactions achieved a threshold level (Ct) of fluorescencewas registered and served to calculate the initial transcript quantityin the RT reaction. Control PCR reactions were effected on the same RTsample, using PCR primers specific to the house keeping gene ribosomalprotein S27a (RPS27A). An amplicon fragment thereof (SEQ ID NO: 23) wasgenerated using the primers set forth in SEQ ID NOs: 21 and 22. For eachprimer set, the value of each PCR reaction was divided into the value ofone of the RTs (a pool of normal lung transcripts from BioChain). Inorder to normalize the results, the ratio for each PCR reaction was thendivided by the house keeping gene ratio for the same RT.

RT PCR for Examples 6-9—1 μg of treated RNA was mixed with 150 ng RandomHexamer primers (Invitrogen) and 500 μM dNTP in total volume of 15.6 μl.The mixture was incubated for 5 min at 65° C. and then quickly chilledon ice. Then, 5 μl 5× SuperscriptII first strand buffer (Invitrogen),2.4 μl 0.1 M DTT and 40 units Rnasin (Promega) were added, and themixture was incubated for 10 min at 25° C., followed by furtherincubation at 42° C. for 2 min. Then, 1 μl (200 units) of SuperscriptII(Invitrogen) was added and the reaction (final volume of 25 μl) wasincubated for 50 min at 42° C. and then inactivated at 70° C. for 15min. The resulting cDNA was diluted 1:20 in 10 mM T0.1E.

Real-Time RT-PCR used for analyzing expression pattern of SEQ ID NOs. 1,9, 18 and 19 in different ovary and lung samples (Examples 7-9, FIGS.8-11)-5 μl of diluted cDNA prepared with random primers were used as atemplate in Real-Time PCR reactions using the SYBR Green I assay (PEApplied Biosystem) with specific primers. The amplification stage waseffected as follows, 50° C. for 2 min, 95° C. for 10 min, 95° C. for 15sec, followed by 60° C. for 1 min. Detection was effected using PEApplied Biosystem SDS 7000. The cycle in which the reactions achieved athreshold level (Ct) of fluorescence was registered and served tocalculate the initial transcript quantity in the RT reaction. Thequantity was calculated using a standard curve created using serialdilutions of either a purified amplicon product or reverse transcription(RT) reaction prepared from RNA mix purified from 5 cell-lines (HCT116,H1299, DU145, MCF7, ES-2). To minimize inherent differences in the RTreaction, the resulting quantity was normalized to the geometric mean ofthe quantities of several housekeeping genes (different HSKP genes wereused for the different tissue panels).

RT-PCR analysis used for analyzing expression pattern of SEQ ID NOs. 18and 19 in different breast samples (examples 10, 11, FIGS. 12,13)-5 μlof diluted cDNA prepared with random primers were used as a template inRT-PCR reactions using the SYBR Green I assay (PE Applied Biosystem)with specific primers. The amplification stage was effected as follows,50° C. for 2 min, 95° C. for 10 min, 95° C. for 15 sec, followed by 60°C. for 1 min. PCR products were analyzed by 1.8% agarose gels.

Example 3 Expression Pattern of a SIM2 Derived Fragment (SEQ ID NO:20)in Normal and Malignant Lung Samples

The expression level of a SIM2 derived fragment corresponding to SEQ IDNO:20, which is a fragment of SEQ ID NO: 1 (nucleotide coordinates125-225, see FIG. 1) was determined using the primers set forth in SEQID NOs: 4 and 5 (designated as primers 1 and 2 in FIG. 1) and measuredby real time PCR. The expression of the housekeeping gene RPS27A(GenBank Accession No. NM_(—)002954, SEQ ID NO: 23) was determinedsimilarly using primers: SEQ ID NOs: 21, 22. Expression values werefirst normalized to a housekeeping gene. The expression was thencalculated relative to a pool of normal lung samples (Lu_(—)6N).

As shown in FIG. 2, representing two duplicates of the same experiment,the expression of the SIM2 fragment (SEQ ID NO: 1) in the normal sampleswas significantly lower than in the tumor samples. Interestingly, highexpression was found in adenocarcinoma samples (4 out of the 9 samples)and squamous cell carcinoma samples (7 out of the 14 samples). Theexpression in these samples was between 10 and 200 fold higher ascompared to the expression pattern in normal samples.

Example 4 Expression Pattern of a SIM2 Derived Fragment (SEQ ID NO:20)in Normal and Malignant Colon Samples

The expression levels of a SIM2 derived fragment corresponding to SEQ IDNO. 20 (a portion of SEQ ID NO: 1 amplified by the primers set forth inSEQ ID NOs. 4-5) as well as the housekeeping gene RPS27A (GenBankAccession No. NM_(—)002954, SEQ ID NO: 23) using primers: SEQ ID NOs:21, 22) were measured by real time PCR. Each value was first normalizedto the housekeeping gene. The expression level was then calculatedrelative to a pool of RNA from normal colon (Col-normal pool).

As shown in FIG. 3, representing two duplicates of the same experiment,the expression in most of the adenocarcinoma and cell lines samples washigher than in the normal samples.

Example 5 Expression Pattern of a SIM2 Derived Fragment (SEQ ID NO: 9)in Normal and Malignant Lung Samples

Expressions of long (SEQ ID NO: 7) and short (SEQ ID NO: 8) SIM variantswas measured by real time PCR using a sequence fragment (SEQ ID NO: 9,and the primers set forth in SEQ ID NOs: 10 and 11, designated asprimers 9 and 10 in FIG. 1, respetively) which is shared by bothsequences. Each expression was first normalized to RPS27A (SEQ ID NO:23; using primers: SEQ ID NOs: 21, 22) (FIGS. 4 through 7). Then theexpression level was calculated relative to a pool of normal lungs(Lu_(—)6N).

As shown in FIGS. 4 through 7, SIM2 expression in normal samples wasvery low. High expression was found in adenocarcinoma samples andsquamous cell carcinoma samples. Note that over-expression in tumorsamples was 10 to 2000 fold relatively to the expression of SIM2 innormal samples. FIGS. 5 and 7 show a better resolution pattern of SIM2expression on a scale of 0-200.

Example 6 Expression of SIM2 Derived Fragments in Normal and CancerousOvary Tissues

The expression of two different SIM2-derived sequences (SEQ ID NOs:20and 9) and three housekeeping genes—PBGD (GenBank Accession No: HSPBGDR,SEQ ID NO: 32; using the primers set forth in SEQ ID NOs: 30, 31),ATP-6-syn (GenBank Accession No: NM_(—)1733702, SEQ ID NO: 26; using theprimers set forth in SEQ ID NOs: 24, 25), 18s ribosomal RNA (GenBankAccession No: HSRRN18S, SEQ ID NO: 29; using the primers set forth inSEQ ID NOs: 27, 28) were measured by real time PCR. In each RT sample,the expression of SIM2 sequences was normalized to the geometric mean ofthe quantities of three housekeeping genes PBGD, ATP-6-syn, 18sribosomal RNA, as detailed in Example 2, hereinabove. The normalizedquantity of each RT sample was then divided by the normalized quantityof a normal sample (No. 45, Table 2 above).

As shown in FIG. 8, SIM2 expression in normal samples (samples nos.42-49, Table 2 above) was significantly lower than in the cancersamples. Notably, the highest expression of SIM2 was found in papilaryserous (carcinoma or adenocarcinoma or cystadenocarcinoma) samples(samples 24, 27, 29, Table 2).

Example 7 Expression of SIM2 Long Variant-Derived Fragment (SEQ IDNO:18) in Normal and Cancerous Ovary Tissues

Expression of SIM2 long variant (GenBank Accession No: gi7108363, SEQ IDNO: 7) was measured by real time PCR using a fragment (SEQ ID NO: 18corresponding to nucleotide coordinates 1551-1670 of SEQ ID NO: 7 usingthe primers set forth in SEQ ID NOs:14-15, designated as primers 12 and13 in FIG. 1, respectively). In addition the expression of twohousekeeping genes—PBGD (GenBank Accession No: HSPBGDR, SEQ ID NO: 32;using the primers set forth in SEQ ID NOs: 30-31) and HPRT1 (GenBankAccession No: GI_(—)32449, SEQ ID NO: 35; using the primers set forthSEQ ID NOs: 33-34), was measured by real time PCR. In each RT sample,the expression of SIM2 sequences was normalized to the geometric mean ofthe quantities of the housekeeping genes as detailed in Example 2,hereinabove. The normalized quantity of each RT sample was then dividedby the averaged quantity of the normal samples (No. 42-48, Table 2).

As shown in FIG. 9, SIM2 expression in normal samples (sample Nos.42-48, Table 2) was significantly lower than in the cancer samples.Notably, the highest expression of SIM2 was found in 4 out of 6papillary serous samples (carcinoma or adenocarcinoma orcystadenocarcinoma).

Example 8 Expression of SIM2Long Variant-Derived Fragment (SEQ ID NO:18)in Normal and Cancerous Lung Tissues

Expression of SIM2 long variant (SEQ ID NO: 7) was measured in normaland cancerous lung tissues (see Table 3, below) by real time PCR using asequence fragment (SEQ ID NO: 18 corresponding to nucleotide coordinates1551-1670 of SEQ ID NO: 7 using the primers set forth in SEQ ID NOs:14-15, designated as primers 12 and 13 in FIG. 1, respectively). Inaddition the expression of three housekeeping genes—SDHA (GenBankAccession No: NM_(—)004168, SEQ ID NO: 38 was measured using the primersset forth in SEQ ID NOs: 36 and 37), RPS27A (GenBank Accession No:NM_(—)002954, SEQ ID NO: 23 was measured using the primers set forth inSEQ ID NOs: 21, 22), PBGD (GenBank Accession No: HSPBGDR, SEQ ID NO: 32;using the primers set forth in SEQ ID NOs: 30, 31), was measured by realtime PCR. In each RT sample, the expression of SIM2 sequences wasnormalized to the geometric mean of the quantities of the housekeepinggenes as detailed in Example 2, hereinabove. The normalized quantity ofeach RT sample was then divided by the averaged quantity of the normalsamples (No. 46-54, Table 3). TABLE 3 Serial number Lot number PathologySource  1 A504117 Adenocarcinoma Biochain  2 A504118 AdenocarcinomaBiochain  3 CG-200 Adenocarcinoma Ichilov  4 Com-02-43T-M-2237TAdenocarcinoma Grade 1 Clinomics  5 Com-02-49T-M-2214T AdenocarcinomaGrade 2 Clinomics  6 Com-02-55T-M-2206T Adenocarcinoma Grade 3 Clinomics 7 Com-02-57T-M-2285T Adenocarcinoma Grade 4 Clinomics  8Com-02-59T-M-2261T Adenocarcinoma Grade 2 Clinomics  9Com-02-41T-M-2269T Adenocarcinoma Grade 3 Clinomics 11Com-02-53T-M-2221T Adenocarcinoma Grade 1 Clinomics 12 A504119Moderately adenocarcinoma Biochain 13 A504116 moderately to poorlyadenocarcinoma Biochain 14 CG-111 Adenocarcinoma Ichilov 15 CG-244Bronchioloalveolar adenocarcinoma Ichilov 16 A409091 Moderately squamousBiochain 17 A503183 moderately squamous Biochain 18 A503387 moderatelyto poorly squamous Biochain 19 A408175 Squamous Biochain 20 A501121Squamous Biochain 21 A503187 Squamous Biochain 22 A503386 SquamousBiochain 23 CG-109 (1) Squamous Ichilov 24 CG-123 Squamous Ichilov 25CG-204 Squamous Ichilov 26 Com-02-47T-M-2208T Squamous Grade 3 Clinomics27 Com-02-61T-M-2215T Squamous Grade 2 Clinomics 28 Com-02-63T-M-2216TSquamous Grade 3 Clinomics 29 Com-02-65T-M-2239T Squamous Grade 1Clinomics 30 A501389 Small cell Biochain 31 A501390 Small cell Biochain32 A501391 Small cell Biochain 33 A504115 Small cell Biochain 34Com-02-45T-M-2217T Small cell Grade 2 Clinomics 35 Com-02-69T-M-2210TSmall cell Grade 3 Clinomics 36 Com-02-71T-M-2218T Small cell Grade 2Clinomics 37 Com-02-73T-M-2235T Small cell Grade 1 Clinomics 38 A504113large cell Biochain 39 A504114 large cell Biochain 40 Com-02-75T-M-2212TLarge cell Grade 3 Clinomics 41 Com-02-77T-M-2257T Large cell Grade 4Clinomics 42 Com-02-79T-M-2241T Large cell Grade 2 Clinomics 43Com-02-163T-M-2290T Large cell Grade 1 Clinomics 44 A501123 Moderatelyalveolus carcinoma Biochain 45 A501221 Alveolus carcinoma Biochain 46A501124 Normal Biochain 47 A503205 Normal Biochain 48 A503206 NormalBiochain 49 A503384 Normal Biochain 51 Com-02-44N-M-2237N normal M4Clinomics 53 Com-02-42N-M-2269N normal M9 Clinomics 54Com-02-48N-M-2208N normal M26 Clinomics

As shown in FIG. 10, SIM2 expression in normal samples (sample Nos.46-54, Table 3) was significantly lower than in the cancer samples.Interestingly, high expression was found in adenocarcinoma samples (7out of the 15 samples) and squamous cell carcinoma samples (9 out of the14 samples).

Example 9 Expression of SIM2Short Variant-Derived Fragment (SEQ IDNO:19) in Normal and Cancerous Ovary Tissues

Expression of SIM2 short variant (GenBank Accession No: gi7108361, SEQID NO: 8) was measured by real time PCR using the sequence fragment setforth in SEQ ID NO: 19 (nucleotide coordinates 1224-2324 of SEQ ID NO:7, amplified using the primers set forth in SEQ ID NOs: 16-17 designatedas primers 14 and 15 of FIG. 1, respectively). In addition theexpression of two housekeeping genes—PBGD was measured by real time PCRas described above. In each RT sample, the expression of SIM2 sequenceswas normalized to the geometric mean of the quantities of thehousekeeping genes as detailed in Example 2, hereinabove. The normalizedquantity of each RT sample was then divided by the averaged quantity ofthe normal samples (No. 42-49, Table 2 above).

As shown in FIG. 11, SIM2 expression in normal samples (sample Nos.42-49, Table 2 above) was significantly lower than in the cancersamples.

Example 10 Expression of SIM2 Long Variant-Derived Fragment (SEQ IDNO:18) in Normal and Cancerous Breast Tissues

Expression of SIM42 short variant (SEQ ID NO: 7) was evaluated by RT-PCRusing a sequence fragment (SEQ ID NO: 18, described above). As shown inFIG. 12, SIM2 expression in most tumor samples was higher than in thenormal samples (Table 4, below).

Example 11 Expression of SIM2 Short Variant-Derived Fragment (SEQ IDNO:19) in Normal and Cancerous Breast Tissues

Expression of SIM2 short variant (SEQ ID NO: 8) was evaluated by RT-PCRof a sequence fragment (SEQ ID NO: 19, primers 14 and 15, see FIG. 1)normal and cancerous breast samples (see Table 4, below) TABLE 4 Serialnumber Cat No. Pathology Source  1 M-0140T DCIS Grade 1 clinomics  2M-0110T DCIS Grade 2 clinomics  3 M-0150T IDC Grade 1 clinomics  4M-2159T IDC Grade 1 clinomics  5 M-2168T IDC Grade 1 clinomics  6 7238TIDC - G1 ABS  7 7263T IDC - G2 ABS  8 M-0113T IDC Grade 2 clinomics  9M-0160T IDC Grade 2 clinomics 10 M-2160T IDC Grade 2 clinomics 11M-2175T IDC Grade 2 clinomics 12 1432T IDC - G2 ABS 13 A0133T IDC - G2ABS 14 A0135T IDC - G2 ABS 15 7259T IDC - G2 ABS 16 20032T IDC - G2 ABS17 20036T IDC - G2(3) ABS 18 M-2169T IDC Grade 2 clinomics 19 M-2162TIDC Grade 2 clinomics 20 M-0111T IDC Grade 3 clinomics 21 M-0112T IDCGrade 3 clinomics 22 M-0114T IDC Grade 3 clinomics 23 M-0115T IDC Grade3 clinomics 24 M-0180T IDC Grade 3 clinomics 25 M-2176T IDC Grade 3clinomics 26 7249T IDC - G3 ABS 27 20072T IDC - G3 ABS 28 M-2161T IDCGrade 3 clinomics 29 M-2170T IDC Grade 3 clinomics 30 M-2177T IDC Grade3 clinomics 31 CG-154 IDC Ichilov 32 7116T Mucinous carcinoma ABS 33M-0140N normal matched to 1T clinomics 34 M-0110N normal matched to 2Tclinomics 35 7238N normal matched to 6T ABS 36 7263N normal matched to7T ABS 37 M-0150N normal matched to 3T clinomics 38 7116N normal matchedto 32T ABS 39 7259N normal matched to 15T ABS 40 1432N normal matched to12T ABS 41 7249N normal matched to 26T ABS 42 20031T IDC Grade 3 ABSIDC = Invasive Ductal CarcinomaDCIS = Ductal Carcinoma In Situ

As shown in FIG. 13, SIM2 expression in normal samples (sample Nos.33-41, Table 4 above) was significantly lower than in the cancersamples.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A method of diagnosing predisposition to, or presence of ovariancancer, breast cancer and/or lung cancer in a subject, the methodcomprising determining a level of SIM2 in a biological sample obtainedfrom the subject, said level being correlatable with predisposition to,or presence or absence of the ovarian cancer, breast cancer and/or lungcancer, thereby diagnosing predisposition to, or presence of ovariancancer, breast cancer and/or lung cancer in the subject, said level ofSIM2 being determined according to expression of a polynucleotidesequence according to SEQ ID NO:7 or a fragment thereof.
 2. The methodof claim 1, wherein said biological sample is a tissue sample and/or abody fluid sample.
 3. The method of claim 2, wherein said tissue sampleis selected from the group consisting of an ovarian tissue, a lungtissue and a breast tissue.
 4. The method of claim 1, wherein said SIM2fragment comprises SEQ ID NOs:
 9. 5. The method of claim 1, wherein saiddetermining level of said SIM2 is effected at an mRNA level.
 6. Themethod of claim 1, wherein said determining level of said SIM2 iseffected at a protein level. 7-17. (canceled)
 18. Use of a SIM42detecting agent for detecting ovarian, breast and/or lung cancer. 19.The use of claim 18, wherein said agent for detecting ovarian, breastand/or lung cancer is an oligonucleotide.
 20. (canceled)
 21. The use ofclaim 18, wherein said agent for detecting ovarian, breast and/or lungcancer is coupled to a detectable moiety selected from the groupconsisting of a chromogenic moiety, a fluorogenic moiety, a radioactivemoiety and a light-emitting moiety. 22-41. (canceled)
 42. The method ofclaim 5, wherein said determining level of said SIM2 comprisesamplification with a primer pair for specifically amplifying apolynucleotide according to SEQ ID NO:7.
 43. The method of claim 42,wherein said primer pair is capable of specifically amplifyingpolynucleotide according to SEQ ID NO:9.
 44. The method of claim 43,wherein said primer pair comprises SEQ ID NOs: 10 and
 11. 45. The methodof claim 1, wherein said lung cancer comprises one of adenocarcinoma orsquamous cell carcinoma.
 46. The method of claim 1, wherein said ovariancancer comprises a papillary serous ovarian cancer.
 47. The method ofclaim 46, wherein said papillary serous ovarian cancer comprises one ofcarcinoma, adenocarcinoma or cystadenocarcinoma.